The laboratory has been focused during the last year on immune-mediated tumor cell destruction. Natural killer (NK) cells and cytotoxic T cells (CTL) lyse virally-infected and tumor cells by 2 main mechanisms:(1) the exocytosis of lytic granules and (2) the expression of cytotoxic molecules like tumor necrosis factor (TNF) or related molecules like Fas-ligand. We have investigated the importance of these mechanisms in cytotoxic immune responses to a murine renal cancer Renca. Using activated T cells from mutant or gene-targeted mice as effector cells, we could demonstrate the lysis of various murine cancer cells in long-term cytotoxicity assays. T cells exclusively used Fas ligand or perforin/granzymes to lyse mouse renal cancer cells. Furthermore, the lysis of the Renca tumor by Fas ligand was not blocked effectively by caspase inhibitors. This is unusual, so the molecular mechanism of tumor cell lysis is currently under investigation. Furthermore, we successfully isolated mouse T cells which specifically killed Renca tumor cells. These specific anti-Renca T cells showed some therapeutic efficacy when transfered to tumor bearing mice in vivo. The cytolytic granules of cytotoxic lymphocytes contain a variety of unique proteins, including a pore-forming protein (perforin) and a family of serine protease enzymes called granzymes. We have further studied the expression of granzyme M (met-ase), a novel granzyme that we had previously cloned. Using a monoclonal antibody specific for granzyme M we have detected this granzyme in freshly isolated NK cells, NKT cells and T gamma delta cells by western blotting and immunohistochemistry. T alpha beta cells do not seem to express this granzyme even after activation. The expression pattern of Granzyme M contrasts with that of the other granzymes and perforin, which can be easily detected in activated T alpha beta cells. This suggests that Granzyme M may be involved in innate rather than adaptive immunity. We are trying to define the biological role of this enzyme, and will soon be obtaining mice where granzyme M has been ablated by gene targeting.